The long term objective of this research effort is to elucidate the molecular mechanisms by which bacteriophage accomplish lysis of the host. Phage lysis is a fundamental phenomenon involving molecules which interact with the host cell membrane and with bacterial cell wall structures. Moreover, the process of host lysis must be carefully scheduled by the bacteriophage in order to optimize the production of virions. Thus lessons learned from the study of phage lysis are likely to be important to the understanding of cell membrane structure and dynamics and the regulation of viral proliferation in general, as well as providing insights into the structure, function, and metabolism of the bacterial envelope. Phage systems provide powerful genetic tools, which we propose to use in a study of the lysis systems of lambda and related phage and of the phage phiX174, representing the two fundamental strategies of host lysis. We will conduct a genetic analysis of the lambda S gene, encoding the prototype "holin", which functions to allow passage of the soluble endolysin, or lysozyme, across the cell membrane. Our approach uses positive and negative selections for holin function using plasmid and phage vectors. A mutational study of a functional homolog from phage 21 will be conducted to determine what features of the 21 holin are common with the lambda holin. Intragenic suppression and complementation will be used to establish a point-to-point interaction-map within the holin sequence. Clock mutants defective in the timing of lysis will be isolated and characterized. An in vitro hole forming assay will be developed using planar lipid membranes or small unilamellar vesicles. The interaction between the phiX174 E lysis protein and the host protein SlyD, which is a homolog of the human FKBP immunophilin, will be investigated using genetic and molecular means.